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U.S.A."],"published-print":{"date-parts":[[2025,4]]},"abstract":"<jats:p>There is considerable interest in the targeted degradation of proteins implicated in human disease. The use of sequence-specific proteases for this purpose is severely limited by the difficulty in engineering the numerous enzyme\u2013substrate interactions required to yield highly selective proteases while maintaining catalytic activity. Herein, we report a strategy to evolve a protease for the programmed degradation of \u03b1-Synuclein, a presynaptic protein closely linked to Parkinson\u2019s disease. Our structure-guided evolution campaign uses the protease from botulinum neurotoxin and showcases the stepwise change of specificity from its native substrate SNAP25 to the selective degradation of \u03b1-Synuclein. The protease\u2019s selectivity is further demonstrated in human cells where near complete degradation of overexpressed human \u03b1-Synuclein is observed with no significant effects on cell proliferation. This stepwise strategy may serve as a general approach to evolve highly selective proteases targeting dysregulated proteins.<\/jats:p>","DOI":"10.1073\/pnas.2426745122","type":"journal-article","created":{"date-parts":[[2025,3,24]],"date-time":"2025-03-24T15:13:45Z","timestamp":1742829225000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":7,"title":["Targeted degradation of \u03b1-Synuclein using an evolved botulinum toxin protease"],"prefix":"10.1073","volume":"122","author":[{"given":"Philipp","family":"Sondermann","sequence":"first","affiliation":[{"name":"Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute","place":["La Jolla"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian S.","family":"Diercks","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute","place":["La Jolla"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia","family":"Rong","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute","place":["La Jolla"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3188-1202","authenticated-orcid":false,"given":"Peter G.","family":"Schultz","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute","place":["La Jolla"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2025,3,24]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1136\/jnnp.2003.034702"},{"key":"e_1_3_4_2_2","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.parkreldis.2006.06.002","article-title":"Botulinum toxin: Clinical use","volume":"12","author":"Truong D. 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